Glycosyltransferase-catalyzed reactions have gained increasing attention and application for the synthesis of complex carbohydrates and glycoconjugates. Most mammalian glycosyltransferases suffer from no or low expression in E. coli systems and more restricted substrate specificity. In comparison, bacterial glycosyltransferases are generally easier to access using E. coli expression systems and have more promiscuous substrate flexibility. Nevertheless, despite the discovery of many bacterial glycosyltransferases which have promiscuities for both donor and acceptor substrates, the application of glycosyltransferases in the synthesis of carbohydrate-containing structures is limited by the availability and the substrate specificity of wild-type enzymes.
For example, sialyltransferases, the key enzymes that catalyze the transfer of a sialic acid residue from cytidine 5′-monophosphate-sialic acid (CMP-sialic acid) to an acceptor, have been commonly used for the synthesis of sialic acid-containing structures. Sialyl Lewisx [SLex, Siaα2-3Galβ1-4(Fucα1-3)GlcNAcβOR] is an important carbohydrate epitope involved in inflammation as well as adhesion and metastasis of cancer cells. It is a well-known tumor-associated carbohydrate antigen and has been used as a candidate for cancer vaccine. The biosynthesis of SLex involves the formation of Siaα2-3Galβ1-4GlcNAcβOR catalyzed by an α2-3-sialyltransferase followed by an α1-3-fucosyltransferase-catalyzed fucosylation. This biosynthetic sequence usually cannot be altered as common α2-3-sialyltransferases do not use fucose-containing Lewisx [Lex, Galβ1-4(Fucα1-3)GlcNAcβOR] as a substrate.
As common terminal monosaccharides, sialic acids constitute a family of great structural diversity. So far, more than 50 structurally distinct sialic acid forms have been identified in nature. To obtain SLex with different sialic acid forms to elucidate the biological significance of naturally occurring sialic acid modifications, an efficient enzymatic approach is to use Lex [Galβ1-4(Fucα1-3)GlcNAcβOR] as a fucose-containing acceptor to add different sialic acid forms by a suitable α2-3-sialyltransferase. This process of introducing different forms of sialic acid onto the common fucosylated acceptor Lex in the last step has significant advantages compared to the normal SLex biosynthetic pathway in which fucosylation is the last glycosylation process. It not only simplifies the synthetic scheme as a less number of reactions are needed, but also makes the purification process much easier as negatively charged SLex product is separated from neutral Lex oligosaccharide instead of separating both negatively charged oligosaccharides SLex and non-fucosylated sialosides if fucosylation occurs in the last step.
We and others have demonstrated that a myxoma virus α2-3-sialyltransferase can use Lex as an acceptor substrate for synthesizing SLex. Nevertheless, the low expression level of the enzyme in E. coli (<0.1 mg L−1 culture) limits its application in preparative and large-scale synthesis of SLex.
We have previously shown that a multifunctional α2-3-sialyltransferase from Pasteurella multocida (PmST1) has a good expression level in E. coli (100 mg L−1 culture) (J. Am. Chem. Soc. 2005, 127, 17618-17619.). It can use Lex as an acceptor for the synthesis of SLex but the yields are poor (<20%) in spite of different conditions tested. What is needed, therefore, are α2-3-sialyltransferases having good α2-3-sialyltransferase activity with good expression levels, and lowered α2-3-sialidase or donor substrate hydrolysis activity. Surprisingly, the present invention meets this and other needs.